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(c)
Calculate the concentration of the apocarotenal internal standard (C
A
), in μg/100 mL, in the
working solution:
C
A
= W
A
x CP
A
x (1000/100)
Where W
A
= the weight of apocarotenal used to make the stock solution, in mg
CP
A
= the chromatographic purity of apocarotenal
1000 = the conversion of mg to μg
100 = the factor for the dilution of 1 mL stock in 100 mL working solution
(d)
Plot the relative responses for each analyte (e.g. RR
Lut
) vs. relative concentration (e.g. RC
Lut
) in
the five calibration solutions
RR
Lut
= (A
Lut
/A
A
)
Where A
Lut
= the peak area of all-
trans
lutein in the calibration solution (AU)
A
A
= the peak area of apocarotenal in the calibration solution (AU)
RC
Lut
= (C
Lut
/C
A
)
Where C
Lut
= the concentration of all-trans-lutein in the calibration solution (μg/100 mL)
C
A
= the concentration of apocarotenal in the calibration solution (μg/100 mL)
Determine the slope of the curve (response factor [RF]) for each carotenoid based on linear
regression, forcing the intercept through zero.
(e)
Calculate the mass of apocarotenal (M
A
), in μg, added to the test samples:
M
A
= (C
A
x V
A
) x (4/50)
Where C
A
= the concentration of apocarotenal in the intermediate or WS (μg/100 mL)
V
A
= the volume of ISTD solution added to each sample, in mL
4 = the volume of apocarotenal intermediate or WS used in the ISTD solution, in mL
50 = the total volume of ISTD solution made, in mL
(f)
Calculate the contents of all-
trans
lutein,
cis
isomers of lutein, and total lutein in the test
samples:
Lut
trans
= (M
A
/M
S
) x (A
Lut
/A
A
) x (100/RF
Lut
)
Where Lut
trans
= the all-
trans
lutein in the sample (μg/100 g)
M
A
= the mass of apocarotenal added to the test sample, in μg
METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE